Paenibacillus terrae mutant as well as application and high-throughput screening method thereof

Abstract

The invention relates to the technical field of microorganisms, and provides a mutant strain of paenibacillus terrae as well as application and a high-throughput screening method of the mutant strain. The mutant strain is named as KY1643, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.33062. According to the invention, a mutant strain of which the algicidal activity and the fungicidal activity are remarkably improved compared with those of a wild type strain is efficiently screened out through a self-established efficient algicidal activity evaluation system, and the mutant strain KY1643 has excellent performance and good agricultural application value. According to the screening method, high-throughput screening of the plant disease control strains can be achieved, and compared with a conventional strain screening method, the screening efficiency is high, and the screening time is greatly shortened.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and specifically, to a mutant of Paenibacillus terrae and its uses and high-throughput screening method. Background Art

[0002] Plant diseases, as one of the important limiting factors in agricultural production, seriously threaten global agriculture and food safety. For a long time, chemical pesticides have been widely used in the prevention and control of plant diseases due to their advantages such as convenience, high efficiency, and low cost. However, this has also led to problems such as environmental pollution, pesticide residues, and disease resistance (Wen Jianjun, 2022). People are urgently hoping to find an alternative method for the prevention and control of plant diseases. In this context, microbial biocontrol has come into the public eye. Using microbial pesticides made from microorganisms and their metabolites for the prevention and control of plant diseases has become one of the research focuses in the field of biocontrol (Zhang Yixian, 2024). The activities of microbial pesticides are mainly bacteria, fungi, viruses, etc., which have advantages such as environmental friendliness, non-toxic side effects, and sustainability. At the same time, they can also enhance the resistance of plants and achieve ideal disease prevention and control effects (Lin Manglu, 2023; Xie Jingyuan et al., 2021). However, traditional microbial control directly uses live biocontrol bacteria, which also exposes certain drawbacks in field control. On the one hand, since most biocontrol microorganisms are obtained through indoor screening, when applied in the field environment, due to the influence of multiple factors (pH, soil type, temperature, etc.) on their survival and colonization, the disease prevention effect may be unstable or lower than their in vitro and indoor control effects (Chen Zhiyi et al., 2002; Jiang Xu, 2018). On the other hand, the yield of bactericidal active substances of wild-type strains isolated from nature is not high, showing an unsatisfactory control effect (Wang Chao et al., 2017).

[0003] To address the above-mentioned dilemmas in microbial control, some researchers have shifted their focus to enhancing the metabolites of biocontrol bacteria. Antibacterial active substances of biocontrol bacteria refer to active substances produced by biocontrol microorganisms that can inhibit the growth and reproduction of pathogenic bacteria and interfere with their physiological metabolism, thereby rendering the pathogenic bacteria inactive or dead (Tao Zong et al., 2019). The metabolites of biocontrol bacteria are relatively stable in nature, less affected by the environment, and have been successfully applied in production. Currently, hundreds of antibacterial active substances of biocontrol bacteria have been reported. According to their active ingredients and mechanisms of action, they are mainly classified into several categories, including antibiotics, antimicrobial peptides, bacteriostatic enzymes, and other small-molecule substances, such as avermectin, Bacitracin, Gramicidin S, Polymyxin E, and the cyclic lipopeptide Epichlicin (Tan Hongsheng, 2024; Gou Zhongxuan et al., 2022; XIAOTS et al., 2023; RENL et al., 2022). The efficiency and yield of producing antibacterial active substances from wild-type strains isolated from nature are often low, and the production cost is high. Currently, this dilemma is mainly solved through physical mutagenesis, chemical mutagenesis, genetic engineering techniques, fermentation process optimization, and other means. Chemical mutagenesis mainly uses chemical reagents to induce mutations in strains. Commonly used chemical mutagenesis reagents include diethyl sulfate (DES), ethyl methanesulfonate (EMS), and nitrosoguanidine (NTG). The mutagenesis mechanism is that chemical reagents can change the DNA structure and cause mutations. The probability of DNA aberration caused by many chemical mutagenesis reagents is low, they have site specificity, and the proportion of mutations generated is high and the range is large. Moreover, this method does not require special equipment for operation, and the operation process is relatively simple (Wang Jing et al., 2016; Xiong Jianchun, 2010). EMS mutagenesis is an ideal mutagenesis method for constructing large-scale saturated mutant libraries currently (Harloff H J et al., 2012). It is the most widely used and has the best effect in mutagenesis breeding. However, EMS mutagenesis is non-directional and highly random. After EMS mutagenesis treatment, a large number of mutant offspring will be generated. It is very important to select a suitable screening system, which is also a technical difficulty in mutagenesis breeding. Mutagenesis breeding will produce a large number of useless mutants. How to achieve high-throughput screening of mutants that meet different criteria is the key to obtaining target mutants. Therefore, developing an efficient screening method for plant disease control strains to obtain mutant strains with better performance has always been one of the important research directions in microbial control.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The object of the present invention is to provide a mutant strain (Paenibacillus terrae) KY1643 of Paenibacillus terrae, its uses, and a high-throughput screening method.

[0006] To achieve the above object of the present invention, the following technical solutions are specifically adopted:

[0007] In a first aspect, the present invention provides a mutant strain of Paenibacillus terrae, and the mutant strain is Paenibacillus terrae KY1643, which is deposited in the China General Microbiological Culture Collection Center, and the deposit number is: CGMCC No. 33062.

[0008] In a second aspect, the present invention provides a microbial agent, and the microbial agent comprises the aforementioned Paenibacillus terrae KY1643 or fermentation broth, bacterial suspension and / or supernatant. In one embodiment, the bacterial cells are live bacterial cells and / or inactivated bacterial cells. In some embodiments, the above-mentioned strain and microbial agent can be prepared into biological fertilizers or other compositions. In one embodiment, the microbial agent is a solid microbial agent with a total viable count of 0.6 - 2.5×10 10 cfu·g- 1 or a liquid microbial agent with a total viable count of 0.6 - 2.5×10 10 cfu·mL- 1

[0009] The strain of the present invention can be made into a bacterial liquid preparation or a bacterial powder preparation, for example, by extracting or drying the fermentation bacterial liquid of the strain. The microbial agent can be prepared in forms such as liquid, solid powder, etc., such as emulsion or suspending agent. The bacterial liquid preparation or bacterial powder preparation may further comprise carriers commonly used in the preparation of microbial agents.

[0010] In a third aspect, the present invention provides the use of Paenibacillus terrae KY1643 or the aforementioned microbial agent in preventing and controlling plant diseases.

[0011] In one embodiment, the plant diseases include one or more of cotton wilt, watermelon wilt, banana wilt, rice sheath blight, gray mold, apple leaf spot, pear black spot, tomato early blight, cotton verticillium wilt. In some specific embodiments, the plants include crop plants, such as vegetable or food crop plants, etc. According to the above use of the present invention, the present invention also relates to a method for preventing and controlling plant pathogens, and the method comprises using the aforementioned Paenibacillus terrae KY1643 or a microbial agent or biological fertilizer containing Paenibacillus terrae KY1643.

[0012] ​In a fourth aspect, the present invention provides the use of the strain KY1643 or the bacterial agent in algae killing or in the preparation of an algicidal product. In one embodiment, the algae includes cyanobacteria or green algae, preferably Microcystis aeruginosa. According to the above use of the present invention, the present invention also relates to a method for inhibiting the growth of algae or removing algae, the method comprising treating algal bodies with the aforementioned Paenibacillus terrae KY1643 or the bacterial agent containing the strain KY1643 or other conventional products; the algae being Microcystis aeruginosa.

[0013] In a fifth aspect, the present invention provides a high-throughput screening method for a Paenibacillus terrae mutant, the screening method comprising the following steps:

[0014] Selecting algae capable of producing chlorophyll as an indicator to establish an algicidal activity evaluation system;

[0015] Screening out mutant strains with improved algicidal activity relative to the wild-type Paenibacillus terrae through the algicidal activity evaluation system; and

[0016] Testing the ability of the mutant strains with improved algicidal activity to kill phytopathogenic bacteria of plant diseases, and screening out target mutant strains with improved phytopathogenic bacteria killing activity relative to the wild-type Paenibacillus terrae.

[0017] In one embodiment, a functional strain with a broad bactericidal spectrum and strong bactericidal activity is selected as the wild-type strain.

[0018] In one embodiment, the wild-type Paenibacillus terrae is Paenibacillus terrae KY834, which is deposited in the China General Microbiological Culture Collection Center, and the deposit number is: CGMCC No. 24708.

[0019] In one embodiment, the selected wild-type strain is mutagenized to produce strain mutants. Preferably, the mutagenesis is chemical mutagenesis. More preferably, the mutagenesis is carried out using EMS to obtain a large number of mutants.

[0020] In one embodiment, the method includes selecting algae capable of producing chlorophyll as an indicator. Preferably, algae of the Chlorophyta or Cyanophyta are selected as indicators, including but not limited to, for example, Chlorella, Scenedesmus, Chlamydomonas, Volvox, Ulothrix, Spirulina, etc.; preferably, freshwater cyanobacteria are selected as indicators.

[0021] In one embodiment, in the screening method, the mutant strain is a mutant strain obtained by mutagenizing the wild-type Paenibacillus terrae KY834 with EMS; the fast-reproducing alga Microcystis aeruginosa that can produce chlorophyll observable by the naked eye and is sensitive to the wild-type strain is selected as an indicator to construct an algicidal activity evaluation system.

[0022] In one embodiment, the mutant strain is a mutant strain obtained by mutagenizing the wild-type Paenibacillus terrae KY834 with EMS.

[0023] In one embodiment, establishing the algicidal activity evaluation system includes: making the alga into a uniform algal suspension and adding it to a solid medium to make algal-containing screening media with different concentrations. In one embodiment, the method includes making a wild-type plant disease control strain to be tested into a bacterial suspension and adding it to the above-mentioned algal-containing screening media for observation. Further, the concentration corresponding to when the growth of algal cells is inhibited or killed and no visible chlorophyll can be produced is selected as the test concentration, and mutants with improved algicidal activity compared to the wild-type are screened by screening the size and transparency of the transparent algicidal zone.

[0024] In one embodiment, Microcystis aeruginosa is made into a uniform algal suspension and added to a solid medium to make algal-containing screening media with different OD 600 concentration values; the wild-type strain is made into a bacterial suspension and spread on the above-mentioned algal-containing screening media with different OD 600 concentration values for culture, and preferably cultured at 25 °C - 32 °C for 40 - 50 h (for example, light-cultured at 30 °C for 48 h) to establish an algicidal activity evaluation system.

[0025] In one embodiment, the method includes selecting the concentration corresponding to when the growth of algal cells is inhibited or killed and no visible chlorophyll can be produced as the test concentration, so that the algicidal activity evaluation system has a higher sensitivity.

[0026] In one embodiment, the algal concentration with the highest sensitivity of the algicidal activity evaluation system is selected as the test concentration (that is, the concentration at which the algicidal ability of a single strain can be judged). At this concentration, the amount of algicidal active substance in a single colony of the wild-type strain just inhibits the growth of algal cells at a specific concentration or kills them so that no visible chlorophyll can be produced. Based on the algicidal zone produced by the wild-type strain at this time, mutants with significantly larger and more transparent transparent algicidal zones than the wild-type (that is, mutant strains with stronger algicidal activity) are screened.

[0027] In a specific embodiment, when the wild-type Paenibacillus terrae in the method is Paenibacillus terrae KY834, the concentration of Microcystis aeruginosa is adjusted to OD 600 = 0.6 to 0.7 (preferably OD 600 = 0.6), so that the algicidal circle produced by KY834 is of an appropriate size; based on the algicidal circle produced by KY834 at this time, a mutant strain with a significantly improved size and transparency of the transparent algicidal circle compared to KY834 has stronger algicidal activity.

[0028] In one embodiment, the algicidal ability of a single strain is judged by producing a transparent algicidal circle on an algal screening medium, and a strain with improved algicidal activity relative to the wild-type strain is selected as a mutant strain.

[0029] In one embodiment, before screening the strain to be screened using the screening medium, the method further includes mutagenizing the strain to be screened.

[0030] In a specific embodiment, the method further includes a step of further screening the obtained target mutant strain. The screening step includes: performing a liquid algicidal ability test on the screened mutant strain and the wild-type strain, and screening out a mutant strain with stronger algicidal ability than the wild-type.

[0031] In a specific embodiment, the screening step further includes: performing an algicidal ability test after gradient dilution of the active substances of the mutant strain and the wild-type strain, and obtaining a mutant strain with stronger algicidal ability than the wild-type strain.

[0032] In a specific embodiment, the screening step further includes: screening a mutant strain with improved ability to kill phytopathogenic fungi compared to the wild-type strain; preferably, extracting and concentrating the bactericidal active substances of the wild-type strain and the mutant strain, and testing the ability of each strain to kill phytopathogenic fungi by the plate confrontation method, to obtain a target mutant with the same spectrum of pathogenic bacteria killed as the wild-type strain and significantly improved bactericidal activity compared to the wild-type strain.

[0033] In a specific embodiment, the phytopathogenic bacteria are fungi, preferably the pathogens causing one of the following plant diseases: cotton wilt, watermelon wilt, banana wilt, rice sheath blight, gray mold, apple leaf spot, pear black spot, tomato early blight, cotton verticillium wilt.

[0034] Under normal circumstances, among the mutant strains generated by EMS mutagenesis, most of the strains have average functions, poor effects, and may be unstable. For the screening of mutant strains, generally, primary screening is carried out in petri dishes at home and abroad, followed by secondary screening through pot experiments for disease prevention, and then re-screening through field experiments for determination. The screening process is cumbersome, with a large workload and a long test time, and is not applicable to the screening of a large number of strains. Therefore, an efficient screening method for excellent strains / strains is very important. The screening method provided by the present invention can simply and quickly screen out strains with biocontrol potential, and is particularly suitable for the high-throughput screening of Paenibacillus terrae mutants, especially for screening mutant strains with both algicidal activity and plant pathogenicidal activity.

[0035] The screening method provided by the present invention can effectively reduce the workload, is simple and reliable, and is particularly suitable for screening after mutagenesis based on wild-type strains with known functions, and has good development and application potential. The screening method of the present invention can simply, quickly and effectively screen out biocontrol strains with application prospects (such as Paenibacillus terrae mutants).

[0036] The present invention aims to improve the activity of wild-type Paenibacillus terrae KY834 (patent number 202211603064.1) against pathogenic fungi of plant diseases, and uses the chemical mutagen EMS to mutagenize it. By establishing an efficient algicidal activity evaluation system, about 73,000 mutant progenies were screened in a short time, and 9 target mutant strains were obtained; through the algicidal activity test in liquid, 3 mutant strains with significantly improved algicidal ability were obtained, namely: Y21, Y23-1, Y23-3; through the plate confrontation test, it was confirmed that the bactericidal spectra of the 3 mutant strains were consistent with that of the wild-type KY834, and the bactericidal activities of these three mutant strains were improved to varying degrees compared with the wild-type KY834. Among them, the fungicidal activity of the mutant strain Y21 was the most significantly improved compared with the wild-type KY834, and its fungicidal activity was increased by more than 4 times compared with the wild-type. Therefore, Y21 was included in the rare functional microbial strain library of Kangshengyuan (Zhaoqing) Biotechnology Co., Ltd. and renamed Paenibacillus terrae KY1643.

[0037] Preservation information: The Paenibacillus terrae provided in this application, named KY1643, is preserved in the General Microbiological Center of the China Microbial Culture Collection Management Committee. The preservation address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the preservation time is: December 13, 2024, and the preservation number is CGMCC NO. 33062. It was detected as a viable strain by the preservation center on December 13, 2024.

[0038] In addition, the wild-type Paenibacillus terrae involved in this application is Paenibacillus terrae KY834, a publicly disclosed strain that was previously deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No. 24708.

[0039] The beneficial effects of the present invention are as follows: The mutant strain of the present invention has a broad-spectrum effect of killing pathogenic bacteria. Compared with the wild-type strain, its bactericidal activity is significantly improved, and its algicidal activity is more excellent, having good popularization and application value.

[0040] The above-mentioned induction and screening method of the present invention has the advantages of simple operation, short cycle, and high efficiency. It is not only applicable to the Paenibacillus terrae of the present invention but also applicable to other microbial strains with rich secondary metabolites. It can screen a large number of samples in a short time, with high sensitivity and improved screening efficiency. Description of the Drawings

[0041] Figure 1 Algae-killing situation of KY834 before adjusting the algae concentration;

[0042] Figure 2 Algae-killing situation of KY834 after adjusting the algae concentration;

[0043] Figure 3 Target mutant strain screened through the high-efficiency algicidal activity evaluation system;

[0044] Figure 4 Test results of the algae-killing ability of 9 microbial mutant strains in the liquid state;

[0045] Figure 5 Test results of the algae-killing ability of each strain when Microcystis aeruginosa is diluted 5 times;

[0046] Figure 6 Test results of the algae-killing ability of each strain when Microcystis aeruginosa is diluted 10 times;

[0047] Figure 7 Test results of the algae-killing ability of each strain when Microcystis aeruginosa is diluted 100 times;

[0048] Figure 8 Results of testing the ability of wild-type KY834 and mutant strain Y21 to kill phytopathogenic fungi by the plate confrontation method (in the figure are Fusarium wilt of cotton, Fusarium wilt of watermelon, Fusarium oxysporum f. sp. cubense race 1, Fusarium oxysporum f. sp. cubense race 2, Fusarium oxysporum f. sp. cubense race 3, Botrytis cinerea, Rhizoctonia solani of wheat, Rhizoctonia solani of rice, and early blight of tomato in sequence);

[0049] Figure 9Results of testing the ability of wild-type KY834 and mutant strain Y23-1 to kill phytopathogenic fungi by the plate confrontation method (in the figure are Fusarium oxysporum f. sp. vasinfectum, Fusarium oxysporum f. sp. niveum, Fusarium oxysporum f. sp. cubense tropical race 1, Fusarium oxysporum f. sp. cubense subtropical race 2, Fusarium oxysporum f. sp. cubense race 4, Botrytis cinerea, Rhizoctonia solani Kühn, Rhizoctonia solani Kuhn, Rhizoctonia solani, Alternaria solani in sequence);

[0050] Figure 10 Results of testing the suspected ability of wild-type KY834 and mutant strain Y23-3 to kill phytopathogenic fungi by the plate confrontation method (in the figure are Fusarium oxysporum f. sp. vasinfectum, Fusarium oxysporum f. sp. niveum, Fusarium oxysporum f. sp. cubense tropical race 1, Fusarium oxysporum f. sp. cubense subtropical race 2, Fusarium oxysporum f. sp. cubense race 4, Botrytis cinerea, Rhizoctonia solani Kuhn, Alternaria solani in sequence);

[0051] Figure 11 Results of the fungicidal test of the active substances in the acetone layer of the cells of each strain;

[0052] Figure 12 Results of the fungicidal test of the active substances in the acetone layer of the cell suspensions of each strain. Detailed implementation manners

[0053] The following details the specific implementation manners of the present disclosure. It should be understood that the specific implementation manners described herein are only for explaining and interpreting the present disclosure, and are not used to limit the present disclosure. Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the protection scope of the present invention. Unless otherwise specified, various reagents and raw materials used in the present invention are commercially available products or products that can be obtained by well-known methods.

[0054] The method of the present application uses a wild-type strain (Paenibacillus terrae KY834) as the starting strain, and a large number of mutants (about 73,000 mutant offspring) are obtained by EMS mutagenesis. In view of the characteristics of this strain in killing algae and pathogenic bacteria, the present invention independently develops an efficient screening method. Specifically, through the algicidal activity evaluation system, target mutant strains with significantly improved algicidal activity are efficiently screened, and further screening is carried out among these mutant strains, and finally mutants with significantly improved fungicidal activity compared with the wild-type strain are screened out.

[0055] The finally screened Paenibacillus terrae Y21 of the present invention was renamed KY1643, and its algicidal ability was significantly improved. It not only has the same bactericidal spectrum as the wild-type Paenibacillus terrae KY834, but also the fungicidal activity is most significantly improved compared with the wild-type KY834.

[0056] According to the present invention, it should be understood that the strain KY1643 of the present invention and Paenibacillus terrae Y21 are the same mutant strain, and the only difference is that Y21 is a temporary name during the experiment, and KY1643 is its official renamed name.

[0057] According to the present invention, it should be understood that the mycelia, spores and extracellular products of the strain KY1643 of the present invention also correspondingly have strong algolytic activity and algicidal activity.

[0058] According to the present invention, it should be understood that terms such as "algicidal", "algolytic", "algae control", "algae removal" have the same or similar meanings, all referring to killing, removing or significantly inhibiting or controlling the continued growth of algae.

[0059] According to the present invention, it should be understood that "improve" or "increase" means a significant improvement or enhancement in quantity or activity level, such as an increase of more than 10%, or reaching a significant level, or a level that can be clearly distinguished by the naked eye.

[0060] Example

[0061] 1. Establishment of algicidal activity evaluation system

[0062] The microbial strain KY834 is a functional strain with a broad bactericidal spectrum and strong bactericidal activity (patent application number: 202211603064.1). In order to further improve its bactericidal activity and enhance its commercial value, chemical mutagenesis was used in this application to improve its bactericidal activity. The strain obtained by chemical mutagenesis does not belong to genetically engineered microorganisms, and there is no need to consider the problems brought by transgenic organisms, making it easier to commercialize. However, screening for mutants with stronger bactericidal activity through chemical mutagenesis also faces the problem of an extremely large number of mutants to be screened. According to previous experience, this method requires screening about 100,000 populations to obtain the target mutant, and similar work is usually completed by robots in developed countries. Therefore, establishing a high-efficiency screening system is the key to the success of this project.

[0063] We used Microcystis aeruginosa, which has a fast reproduction rate, can produce chlorophyll observable by the naked eye, and is sensitive to the strain KY834, as the indicator of this system to establish a sensitive and efficient screening system. The specific operation is as follows:

[0064] 1.1 Establishment of algicidal activity evaluation system

[0065] (1) Test materials: Paenibacillus terrae KY834 (i.e., the wild-type strain KY834 belongs to a publicly disclosed patented strain (Patent No.: 202211603064.1), obtained from the microbial strain library of Kangshengyuan (Zhaoqing) Biotechnology Co., Ltd.);

[0066] Microcystis aeruginosa: Obtained from the microbial strain library of Kangshengyuan (Zhaoqing) Biotechnology Co., Ltd., and can also be obtained from other commercial channels;

[0067] BG11 solid medium: 1.5 g sodium nitrate, 0.04 g dipotassium hydrogen phosphate trihydrate, 0.075 g magnesium sulfate heptahydrate, 0.036 g calcium chloride dihydrate, 0.006 g citric acid, 0.006 g ferric ammonium citrate, 0.001 g EDTA, 0.02 g sodium carbonate, 0.00286 g boric acid, 0.00181 g manganese chloride monohydrate, 0.000222 g zinc sulfate heptahydrate, 0.000079 g copper sulfate pentahydrate, 0.00039 g sodium molybdate dihydrate, 0.000049 g cobalt nitrate hexahydrate, 1000 mL water, 15 g agar; pH = 7.1, autoclaved at 121.0 °C for 30 min.

[0068] R2A solid medium: 0.25 g peptone, 0.25 g yeast, 0.25 g tryptone, 0.25 g glucose, 0.25 g soluble starch, 0.15 g sodium pyruvate, 0.15 g potassium dihydrogen phosphate, 0.025 g magnesium sulfate, 500 mL H 2 O, 7.5 g agar, autoclaved at 121.0 °C for 30 min.

[0069] (2) Scrape Microcystis aeruginosa from the plate to make a uniform algal suspension;

[0070] (3) Add the above algal suspension to the mixed solid medium of BG11 solid medium and R2A solid medium (volume ratio 1:1) so that the final OD value in the medium is 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 of algae to make an algal-containing screening medium; 600 value of 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 of algae to make an algal-containing screening medium;

[0071] (4) Make a bacterial suspension of the test strain KY834 (wild type), evenly coat it on the algal-containing screening media of each concentration, and place it in a light incubator at 30 °C for 48 hours and then observe;

[0072] (5) Use the system established in the above steps to evaluate the algicidal activity.

[0073] In this system, both the size and transparency of the algicidal clear zone are simultaneously affected by two factors: the concentration of algae and the amount of algicidal active substances. When the concentration of algae and the amount of bactericidal active substances reach a dynamic equilibrium, the amount of bactericidal active substances produced by a single colony is just enough to inhibit the growth of algal cells or cause their death at a specific concentration, and thus no visible chlorophyll can be produced. At this time, the sensitivity of this algicidal activity evaluation system is the highest. Therefore, the algal concentration at which the strain KY834 is in the above situation is selected as the test concentration. Based on the algicidal zone produced by KY834 at this time, mutants with significantly larger and more transparent algicidal clear zones than KY834 have stronger algicidal activity.

[0074] 1.2 Results

[0075] Microcystis aeruginosa can produce visible chlorophyll in a short time. The algicidal activity of the strain KY834 is manifested by the formation of a transparent algicidal clear zone on the algal selective medium. The reason is that the algal cells at this location are inhibited or killed and cannot produce visible chlorophyll, ultimately forming a transparent algicidal clear zone.

[0076] The results are shown in Figure 1 and Figure 2 . Figure 1 To adjust the algicidal situation of KY834 before adjusting the algal concentration, bacterial aggregation and adhesion of the transparent algicidal clear zones occurred on this plate. At this time, it is impossible to accurately judge the strength of the algicidal ability of a single strain. Figure 2 In this experiment, by adjusting the concentration of Microcystis aeruginosa to OD 600 = 0.6, the algicidal zone produced by KY834 is of an appropriate size, and the transparent zone produced is just observable, thus establishing a highly efficient screening system. This highly efficient screening system can solve three situations: First, it avoids the adhesion of adjacent algicidal zones due to too large algicidal zones, which affects the judgment of the algicidal ability of a single strain; Second, strains with algicidal ability weaker than KY834 cannot produce transparent zones, thus eliminating the work of screening such strains; Third, this system can simultaneously plate dozens or even hundreds of colonies on a single plate. As Figure 3 shown, the size and transparency of the transparent zone of the strain in the red circle in the figure are better than those of the strain KY834, and it is the target mutant.

[0077] 2. Obtaining mutants

[0078] 2.1 Materials and reagents

[0079] Paenibacillus terrae KY834: obtained from the microbial strain library of Kangshengyuan (Zhaoqing) Biotechnology Co., Ltd.; Microcystis aeruginosa: obtained from the microbial strain library of Kangshengyuan (Zhaoqing) Biotechnology Co., Ltd.; Ethyl methyl sulfonate (EMS): Shanghai Macklin Biochemical Co., Ltd., with a purity of 99%, batch number: Lot#C16560317; Phosphate buffer (PBS): Shengshi Biomarker (Xiamen) Technology Co., Ltd.

[0080] 2.2 Test methods

[0081] 2.2.1 Activation of bacterial strains and selection of mutagenic bacterial age

[0082] After activating the wild-type strain KY834, inoculate the bacterial strain into R2A liquid medium and culture it at 30 °C and 200 r / min. Take samples at regular intervals and observe the vegetative growth of the bacteria under a 40-fold microscope, and measure the OD 600 value.

[0083] 2.2.2 Preparation of bacterial suspension

[0084] Culture the strain KY834 in R2A liquid medium until the late logarithmic growth phase (no spores are produced). Centrifuge at 6000 r / min for 30 min to collect the precipitate to obtain the bacteria. Make a bacterial suspension with 1 mol / L phosphate buffer (pH = 7.0, sterile), then centrifuge and wash twice, and dilute to 10 9 CFU / mL for standby.

[0085] 2.2.3 Ethyl methyl sulfonate (EMS) mutagenesis: Use phosphate buffer (pH 7.0) to prepare mutagenic agents with final concentrations of 0, 0.1, and 0.2 mol / L from a 1 mol / L EMS stock solution. Filter through a 0.45 μm filter membrane and mix evenly with the bacterial suspension at a ratio of 1:1. Incubate at 30 °C and 200 r / min for 60 min, then take out to obtain treatment solution A. Add 10 mL of 2% sodium thiosulfate solution to every 1 mL of treatment solution A and dilute to 10 -2 ~10 -4 to obtain treatment solution B.

[0086] 2.2.4 Screening of mutants

[0087] Spread the treatment solution B obtained in "2.2.3" on the algae-containing solid medium (OD 600 = 0.6) in "Example 1", and culture it in a light incubator (30 °C) for 48 h. Take it out and observe the size and transparency of the algicidal clear zone, and pick out the strains with large and highly transparent clear zones for further research.

[0088] 2.2.5 Repeated verification

[0089] Inoculate the strain with a large and highly transparent clear zone obtained in "2.2.4" onto the algal-containing solid medium (OD 600 = 0.6) in "Example 1", and use the streak plate method to obtain single colonies. Observe and record the size and transparency of the clear algicidal zones caused by the single colonies, and calculate the algicidal ability coefficient to exclude microorganisms that produce clear algicidal zones with a size and transparency inferior to those of the wild type KY834 on a specific medium, and initially obtain the target mutants with enhanced algicidal ability.

[0090]

[0091] Note: X is the weighted coefficient of transparency, which are 0, 1, and 2 respectively. 0 indicates opaque, 1 indicates that the algicidal zone produced by the bacteria is semi-transparent, and 2 indicates that the algicidal zone produced by the bacteria is fully transparent.

[0092] 2.3 Results

[0093] 2.3.1 Selection of mutagenized cell age

[0094] According to the microscopic observation results and OD 600 value, when the strain KY834 is cultured for 42 hours, it is in the vegetative state, and spores begin to be produced when the culture time exceeds 42 hours. Therefore, 42 hours is selected as the culture time of the starting strain.

[0095] 2.3.2 Mutagenesis results

[0096] The wild type Paenibacillus terrae KY834 was mutagenized with the chemical mutagen EMS. Through multiple rounds of rescreening by chemical mutagenesis, a total of about 73,000 mutagenized offspring were screened, and initially 32 mutant strains with an increased diameter or enhanced transparency of the algicidal zone were obtained. After further verification on the selective medium, among them, 23 mutant strains with an insignificant increase in the size or transparency of the algicidal clear zone compared to the wild type strain KY834 were found. By calculating the algicidal ability coefficient, it was found that the algicidal ability coefficient of these 23 mutant strains did not increase significantly; in addition, 9 mutant strains with a significantly increased transparency, significantly increased size, and significantly increased algicidal ability coefficient of the algicidal zone were also obtained. The results are shown in Table 1. These 9 mutant strains with a significantly increased transparency, significantly increased size, and significantly increased algicidal ability coefficient of the algicidal zone are mutant strains D1, D2, Y7, Y9, Y14, Y18, Y21, Y23-1, and Y23-3.

[0097] Table 1. Results of mutagenesis screening

[0098]

[0099] Note: The algicidal ability coefficient (E) of the wild-type Paenibacillus terrae KY834 is 5.56.

[0100] 3. Algalicidal ability test of 9 mutant strains with enhanced algicidal ability in liquid

[0101] The algicidal ability tests of the 9 obtained mutant strains with enhanced algicidal ability in liquid were all verified by the algicidal ability on solid plates. Now, the algicidal ability tests of the above 9 positive mutant strains in the liquid state are carried out as follows:

[0102] 3.1 Algalicidal ability test in liquid state using Microcystis aeruginosa

[0103] (1) Take an appropriate amount of the 9 target mutant strains (D1, D2, Y7, Y9, Y14, Y18, Y21, Y23-1, Y23-3) obtained in "2.3" from the -80 °C refrigerator, activate them respectively with R2A solid medium, and culture them at 30 °C for 48 hours;

[0104] (2) Culture the microorganisms cultured in "3.1(1)" respectively with R2A liquid medium at 30 °C and 200 rpm for 4.5 d;

[0105] (3) Filter the bacterial liquid obtained in "3.1(1)" with a bacterial filter membrane (0.45 μm) and retain the filtrate;

[0106] (4) Dilute the above filtrate 5 times to obtain a treatment solution;

[0107] (5) Set water as the blank control, the treatment solution of strain KY834 as the positive control, and the treatment solutions of each microbial strain as the treatment groups. Algalicidal ability tests were carried out respectively when Microcystis aeruginosa (OD 600mm = 0.655) was diluted 5 times and 10 times. Among them, the volume ratio of the algal liquid of Microcystis aeruginosa to the treatment solutions (water, the filtrate of strain KY834 diluted 5 times, the filtrates of each strain diluted 5 times) was 1:1, and they were cultured in a light incubator (30 °C) for 3 days.

[0108] (6) After culturing for 3 days, take them out for observation, and record the algal growth situation in Table 2; use the algal growth grade coefficient to represent the algal growth situation in each treatment group, and use the algal growth situation to measure the algicidal ability of each strain: the more the algae in the micropores are inhibited and the less observable chlorophyll is produced, the smaller the algal growth grade coefficient, the closer the liquid in the micropores is to transparency, and the stronger the algicidal ability of the corresponding strain.

[0109] 3.2 Results

[0110] Using Microcystis aeruginosa, the algicidal ability of the 9 positive mutant strains was tested multiple times in the liquid state. The results are shown in Table 2. Figure 4As shown. From Table 2, Figure 4 It can be seen that in the microplate containing Microcystis aeruginosa, the micro-wells where the strains KY834, D1, D2, Y7, and Y18 are located all show green. Microcystis aeruginosa can produce observable chlorophyll, the growth grades of the algae are all above 3.5, and the growth and reproduction of the algae are less inhibited or not inhibited. Correspondingly, when the corresponding strains (KY834, D1, D2, Y7, Y18) are in the presence of Microcystis aeruginosa at this concentration, their algae-killing ability is weak. In the microplate containing Microcystis aeruginosa, the micro-wells where the strains Y9, Y14, Y21, Y23-1, and Y23-3 are located are all transparent. Microcystis aeruginosa does not produce observable chlorophyll, the growth grades of the algae are all 0, the growth and reproduction of the algae are significantly inhibited or have died. Correspondingly, when the corresponding strains (Y9, Y14, Y21, Y23-1, Y23-3) are in the presence of Microcystis aeruginosa at this concentration, their algae-killing ability is strong, and their algae-killing ability is stronger than that of the wild-type KY834.

[0111] Among them, the 5 microorganisms Y9, Y14, Y21, Y23-1, and Y23-3 have algae-killing ability in liquid. After multiple repeated tests, it is preliminarily judged that the algae-killing ability of the strains Y9, Y14, Y21, Y23-1, and Y23-3 in liquid is stronger than that of the wild-type KY834.

[0112] Table 2. Test results of the algae-killing ability of 9 mutant strains in liquid state (represented by the growth of algae)

[0113]

[0114] Note: The numbers in the table are the algae growth grade coefficients, representing the growth of algae; 4 means no difference in growth from the natural state and not inhibited; the smaller the number, the greater the inhibition, the less observable chlorophyll produced by the algae, and the stronger the algae-killing ability of the corresponding strain; 0 means the algae do not grow, the liquid in the micro-well is transparent, and the algae-killing ability is very strong at this time.

[0115] 4. Comparison of the algae-killing ability of 5 algae-killing mutant strains

[0116] In order to compare the algae-killing ability of the mutant strains (Y9, Y14, Y21, Y23-1, Y23-3) that can kill algae in liquid obtained in "3.2" with that of the wild-type KY834, the active substances produced by the above strains were serially diluted, and the algae-killing ability test was carried out in liquid.

[0117] 4.1 Algae-killing ability test in liquid state

[0118] (1) Appropriately take out the 5 algicidal mutant strains (Y9, Y14, Y21, Y23-1, Y23-3) and the wild type KY834 obtained in "3.2" from the -80°C refrigerator, activate them respectively with R2A solid medium, and culture them at 30°C for 48 hours;

[0119] (2) Culture the well-cultured microorganisms in "4.1(1)" respectively with R2A liquid medium at 30°C and 200 rpm for 4.5 d;

[0120] (3) Filter the bacterial liquid obtained in "4.1(2)" with a bacterial filter membrane (0.45 μm) and retain the filtrate;

[0121] (4) Dilute the above filtrate 5 times, 10 times, 20 times, 40 times, 60 times, and 80 times respectively to obtain treatment solutions;

[0122] (5) Set water as the blank control, the treatment solution of strain KY834 as the positive control, and the treatment solutions of each microbial strain as the treatment groups. Conduct algicidal ability tests under the conditions of Microcystis aeruginosa (OD 600mm = 0.655) diluted 5 times, 10 times, and 100 times. The volume ratio of the algal liquid of Microcystis aeruginosa to the treatment solution is 1:1, and culture in a light incubator (30°C) for 3 days.

[0123] 4.2 Results

[0124] Measure the OD 600 values of the algicidal mutant strains (Y9, Y14, Y21, Y23-1, Y23-3) and the wild type KY834 that can kill algae in liquid, and find that the OD 600 values of the mutant strains (Y9, Y14, Y21, Y23-1, Y23-3) and the wild type KY834 do not differ much, and the growth cycle of the mutant strains has basically not changed; then gradient-dilute the active substances they produce and conduct algicidal ability tests in liquid. The results are shown in Tables 3, Figure 5 、 Figure 6 、 Figure 7 as shown.

[0125] The wild-type KY834 can only inhibit the growth and reproduction of algae or cause them to die without producing observable chlorophyll when its bacterial liquid (active substance) is not diluted. The mutant strains Y9 and Y14 can inhibit the growth and reproduction of algae or cause them to die without producing observable chlorophyll when their bacterial liquids (active substances) are not diluted and diluted 5-fold; when the dilution multiple exceeds 5-fold, their algicidal ability decreases; their algicidal ability is 5 times higher than that of the wild-type KY834. When the bacterial liquid (active substance) of the mutant strain Y21 is diluted 10-fold, it can still inhibit the growth and reproduction of algae or cause them to die without producing observable chlorophyll, and its algicidal ability is 10 times higher than that of the wild-type KY834. When the bacterial liquid (active substance) of the mutant strain Y23-1 is diluted 20-fold, it can still inhibit the growth and reproduction of algae or cause them to die without producing observable chlorophyll, and its algicidal ability is 20 times higher than that of the wild-type KY834. When the bacterial liquid (active substance) of the mutant strain Y23-3 is diluted 5-fold, it can inhibit the growth and reproduction of algae diluted 5-fold or cause them to die without producing observable chlorophyll. When its bacterial liquid (active substance) is diluted 10-fold, it can inhibit the growth and reproduction of algae diluted more than 5-fold or cause them to die without producing observable chlorophyll, and its algicidal ability is 5-10 times higher than that of the wild-type KY834.

[0126] In summary, the algicidal abilities of the mutant strains Y9, Y14, Y21, Y23-1, and Y23-3 that can kill algae in liquid are 5 times, 5 times, 10 times, 20 times, and 5-10 times higher than that of the wild-type KY834, respectively. Among them, the algicidal abilities of the mutant strains Y21, Y23-1, and Y23-3 are significantly improved (see Figure 4 、 5 、6), with strong algicidal activity and can be used as the object of subsequent research.

[0127] Table 3. Test results of the algicidal abilities of mutant strains (Y9, Y14, Y21, Y23-1, Y23-3) and wild-type KY834 in liquid state (represented by the growth of algae)

[0128]

[0129] Note: The numbers in the table are the algal growth grade coefficients, representing the growth of algae; 4 indicates no difference from the growth in the natural state and no inhibition; the smaller the number, the greater the inhibition, the less observable chlorophyll produced by the algae, and the stronger the algicidal ability of the corresponding strain; 0 indicates that the algae do not grow and the liquid in the micropores is transparent, and the algicidal ability is very strong at this time.

[0130] 5. Test the bactericidal ability of mutant strains with significantly improved algicidal activity against phytopathogenic fungi

[0131] The ultimate goal of this application is to obtain mutant strains with enhanced ability to kill phytopathogenic fungi compared to the wild-type KY834. Therefore, the present invention conducted tests on the three mutant strains with significantly improved algicidal ability obtained in "4.2" and the wild-type KY834 for their ability to kill phytopathogenic fungi. The specific steps are as follows:

[0132] 5.1 Testing the ability of each strain to kill phytopathogenic fungi by the plate confrontation method

[0133] Set the pathogens of three specializations of cotton wilt, watermelon wilt, banana wilt, root rot, rice sheath blight, wheat sheath blight, gray mold, tomato early blight, pear black spot, and apple blotch leaf spot as the indicator strains, KY834 as the control strain, and the three excellent mutant strains in "4.2" as the test strains. Punch a 0.5 cm fungal cake from the activated indicator strain and inoculate it onto the R2A solid medium. Inoculate the control strain and the test strain 3 cm away from the fungal cake. Each treatment is repeated 3 times for a plate antagonism test against a broad spectrum of phytopathogenic fungal pathogens. Incubate at 30 °C for 6 days, observe the strength of the antagonism of the strains against the plant pathogens, measure the bactericidal circle and colony radius, and calculate the bactericidal ability coefficient.

[0134]

[0135] 5.2 Comparing the activity of each mutant strain and the wild-type KY834 in killing phytopathogenic fungi

[0136] In "5.1", the ability of each strain to kill phytopathogenic fungi has been qualitatively tested. Now, in order to quantitatively measure the bactericidal activity of each strain, various solvents such as chloroform, ethyl acetate, and 75% acetone were used to extract the bactericidal active substances of the mutant strains and the wild-type KY834. It was found that 75% acetone could extract its bactericidal active substances. Therefore, a microplate was used to test the activity of the acetone active substances in the cell bodies and cell suspensions of each strain against phytopathogenic fungi. The specific steps are as follows:

[0137] (1) Activate the strains KY834, Y21, Y23-1, and Y23-3 from the -80 °C refrigerator using R2A solid medium and incubate at 30 °C for 48 hours;

[0138] (2) Scrape approximately 0.1 g of cell bodies from the plates of the cultured strains KY834, Y21, Y23-1, and Y23-3, extract them with 75% acetone, and centrifuge to obtain the supernatant for use;

[0139] (3) Culture the microorganisms cultured in "5.2(1)" separately in R2A liquid medium at 30 °C and 200 rpm for 4.5 days; extract the cultured cell suspensions with 75% acetone and centrifuge to obtain the supernatant for use;

[0140] (4) Dry the supernatant obtained in "5.2(1) and (2)", and then redissolve it with 20% DMSO to obtain a fungicidal treatment solution concentrated 10 times. Dilute it successively with methanol solution to obtain fungicidal treatment solutions concentrated 5 times, 2.5 times, and 1.25 times for standby use.

[0141] (5) Scrape the pathogenic bacteria of Fusarium oxysporum f. sp. cubense race 2 cultured in a petri dish and place them in 20 mL of sterilized water to make a fungal suspension. Dilute the fungal suspension 20 times for standby use.

[0142] (6) Add the treatment solutions concentrated 10 times, 5 times, 2.5 times, and 1.25 times obtained in "5.2(4)" to a microplate (96 wells) containing solid PDA with chloramphenicol, 30 μL per well, and repeat each treatment four times.

[0143] (7) Then add the fungal suspension diluted 20 times to the 96-well plate containing the treatment solution, 20 μL per well, and culture it at 30 °C for 3 d, then take it out for observation.

[0144] 5.3 Results

[0145] Using the plate confrontation method, test the fungicidal activity of KY834, Y21, Y23-1, and Y23-3 against phytopathogenic fungi. The results show that KY834, Y21, Y23-1, and Y23-3 have bactericidal effects on 14 kinds of phytopathogenic fungi (as shown in Table 4).

[0146] Table 4. Bactericidal effects of wild-type KY834 and mutant strains Y21, Y23-1, and Y23-3 against 14 kinds of phytopathogenic fungi (expressed by bactericidal ability coefficient)

[0147]

[0148] Among them, the test results of the fungicidal activity against several important plant diseases are as Figures 8 to 10 shown. As Figure 11 shown, use 75% acetone to extract the active substances of the mycelia of strains KY834, Y21, Y23-1, and Y23-3, and use a microplate to test the fungicidal activity against phytopathogenic fungi. The results show that after the active substances in the acetone layer of wild-type KY834 and mutant strains Y21, Y23-1, and Y23-3 with strong algicidal activity are concentrated 10 times, 1.25 - 2.5 times, 2.5 - 5 times, and 10 times respectively, the growth of fungi in the corresponding micro-wells begins to be inhibited, manifested as no fungi growing in multiple micro-wells or the reduction of the fungal growth area. Compared with wild-type KY834, the fungicidal activities of mutant strains Y21, Y23-1, and Y23-3 with strong algicidal activity are increased by more than 4 times, 2 - 4 times, and 1 time respectively. As Figure 12As shown in the figure, 75% acetone was used to extract the active substances from the bacterial suspensions of strains KY834, Y21, Y23-1, and Y23-3, and a microplate was used to test the activity against phytopathogenic fungi. The results showed that after concentrating the active substances in the acetone layer of the wild-type KY834 by 5 times, only two microwells grew fungi, indicating that the growth of fungi began to be inhibited; after concentrating the active substances in the acetone layer of the mutants Y21 and Y23-1 with strong algicidal activity by 1.25 times, no fungi grew in the microwells where they were located; after concentrating the active substances in the acetone layer of the mutant Y23-3 with strong algicidal activity by 1.25 times, only two microwells grew fungi and the growth area of the fungi decreased, indicating that the growth of fungi began to be inhibited. Compared with the wild-type KY834, the fungicidal activities of the mutants Y21, Y23-1, and Y23-3 with strong algicidal activity were increased by more than 4 times, more than 4 times, and 4 times, respectively.

[0149] In summary, after chemical mutagenesis, the bactericidal spectra of the mutants Y21, Y23-1, and Y23-3 with strong algicidal activity were consistent with that of the wild-type KY834, and the corresponding broad-spectrum properties remained unchanged; the active substances of the wild-type KY834 and the three mutants Y21, Y23-1, and Y23-3 were extracted and concentrated, and combined with the activity test against phytopathogenic fungi, it was found that the fungicidal activities of the three mutants were increased by more than 4 times, 2-4 times, and 1 time, respectively, compared with the wild-type; the active substances of the wild-type KY834 and the three mutants Y21, Y23-1, and Y23-3 were extracted and concentrated, and combined with the activity test against phytopathogenic fungi, it was found that the fungicidal activities of the three mutants were increased by more than 4 times, 2-4 times, and 1 time, respectively, compared with the wild-type. Based on the test results of the three aspects, the mutant Y21 had a broad bactericidal spectrum, and its bactericidal activity was increased by more than 4 times compared with the wild-type KY834, showing the best performance.

[0150] 6 Conclusions

[0151] In the present invention, the wild-type Geobacillus terrestris KY834 was chemically mutagenized, and about 73,000 mutant progenies were screened in a short time through the established high-efficiency algicidal activity evaluation system, and 9 mutants were obtained; through the algicidal activity test and comparison in liquid, 3 mutants with significantly improved algicidal ability were obtained, namely: Y21, Y23-1, and Y23-3; through the plate confrontation test, it was confirmed that the bactericidal spectra of the 3 mutants were consistent with that of the wild-type KY834, and the bactericidal activities of these three mutants were improved to varying degrees compared with the wild-type KY834. Among them, the fungicidal activity of the mutant Y21 was improved most significantly compared with the wild-type KY834, and its fungicidal activity was increased by more than 4 times compared with the wild-type. Among them, the strain Y21 with the best performance was renamed KY1643 (which has been deposited in the China General Microbiological Culture Collection Center, and the deposit number is CGMCC No. 33062).

Claims

1. A mutant strain of Paenibacillus terrestris, characterized in that: The mutant strain is Paenibacillus terrae KY1643, which is deposited in the General Microbiological Center of China Association for Microbiological Culture Collection, with a deposit number of CGMCC No.33062.

2. A bacterial agent, characterized in that The bacterial agent comprises the bacterial body or fermentation liquid, bacterial suspension and / or supernatant of Paenibacillus terrae KY1643 according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacteria are live bacteria and / or inactivated bacteria.

4. Use of Paenibacillus terrae KY1643 according to claim 1 or the bacterial agent according to claim 2 or claim 3 in preventing and controlling plant diseases.

5. The use according to claim 4, characterized in that The plant diseases include one or more of cotton wilt, watermelon wilt, banana wilt, rice sheath blight, gray mold, apple leaf spot, pear black spot, tomato early blight, and cotton verticillium wilt.

6. Use of Paenibacillus terrae KY1643 according to claim 1 or the bacterial agent according to claim 2 or claim 3 in killing algae or preparing algae-lysing products.

7. The use according to claim 6, characterized in that The algae include Microcystis aeruginosa.

8. A high-throughput screening method for Paenibacillus terrestris mutants, the screening method comprising the following steps: Algae that can produce chlorophyll were selected as indicators to establish an algicidal activity evaluation system; Screening out mutant strains with improved algicidal activity compared to wild-type Paenibacillus terrestris by using the algicidal activity evaluation system; as well as The mutant strain with improved algaecidal activity is tested for its ability to kill plant pathogens, and a target mutant strain with improved fungicidal activity against plant pathogens compared with wild-type Paenibacillus terrestris is screened out.

9. The screening method according to claim 8, wherein: The wild type Paenibacillus terrae is Paenibacillus terrae KY834, which is deposited in the General Microbiological Center of China Association for the Collection of Microbiological Cultures, with a deposit number of CGMCC No.24708.

10. The screening method according to claim 9, wherein the screening method uses Microcystis aeruginosa as an indicator, and the mutant strain is a mutant strain obtained by using the wild type of Paenibacillus terrae (Paenibacillus terrae) KY834 as a starting strain and using EMS to induce mutagenesis.

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